Malignant melanoma is a significant and deadly form of cancer worldwide. Emerging evidence has shown that melanoma cells express tumor-associated antigens that are specifically recognized by the immune system. Melanoma antigen-specific T-lymphocytes can be isolated from the tumors of late stage cancer patients or induced by active vaccination using well-characterized tumor peptide epitope vaccines. Adoptive transfer therapy of autologous tumor infiltrating lymphocytes (TILs) has been shown to mediate durable regression of malignant melanoma in particular patients. Furthermore, ex vivo antigen-specific expansion of autologous T-cells from vaccinated patients is a promising approach to increase the potency and frequency of tumor antigen specific T-cells for adoptive immunotherapy. However, current culture methods for ex vivo lymphocyte expansion to produce billions of T-cells for patient therapy are costly, labor intensive and consist of multiple manual open-process steps which are difficult to implement for wider patient delivery without specialized facilities under increasingly stringent regulatory requirements. In addition, prolonged culture of T-cells leads to replicative senescence with loss of biological function and therapeutic activity. Aastrom Biosciences, Inc. is developing a novel clinical scale bioreactor system for production of cells for human cell therapy using closed system automation and continuous single-pass perfusion technologies. The primary goal of this Phase I proposal is to demonstrate the feasibility of using the AastromReplicell/TM Cell Production System to expand highly active melanoma tumor antigen specific T-lymphocytes from patients' tumors or apheresis cells of vaccinated donors for immunotherapy against malignant melanoma. Multiple culture parameters for both antigen-independent and selective melanoma peptide driven T-cell expansion processes will be implemented and evaluated in the clinical scale bioreactor system. The beneficial effect of single-pass perfusion for potentially improved biological function and replicative capabilities of T-lymphocytes when compared to conventional methods will be defined. A closed automated bioreactor system will fulfill a large unmet clinical demand for consistent, reliable and reproducible T-cell production under stringent regulatory conditions with improved immunologic and therapeutic potency for immunotherapy of cancer.